Testing of SNS-032 in a panel of human neuroblastoma cell lines with acquired resistance to a broad range of drugs

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclindependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3rCDDP1000 in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition–mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases

BAX and BAK proteins are required for cyclin-dependent kinase inhibitory drugs to cause apoptosis

In previous reports we have shown in SH-SY5 cells that Olomoucine and Roscovitine, two inhibitory drugs of Cyclin Dependent Kinases, caused apoptosis independent of the extrinsic pathway. In this experimental paradigm, apoptosis was refractory to the protective effects of either Bcl-2 or Bcl-XL overexpression. We are now reporting the failure of Bcl-XL to prevent dell death was consistent with no effect on the kinetics of caspase activation and cytochrome c release. To further characterize this issue, we have discarded a direct effect of either Olomoucine or Roscovitine on mitochondrial permeability transition. Moreover, we have evidence that an intrinsic pathway took place in SH-SY5Y cells by demonstrating the mitochondrial translocation of a GFP-Bax construct upon transfection and treatment with CDK inhibitory drugs. Finally, we tested the effect of Olomoucine and Roscovitine on w.t., bax-/-, bak-/- and double bax-/-bak-/- MEFs. In w.t. MEFs, both drugs induced cell death by apoptosis in a dose-dependent manner. In bax-/-, bak-/- and, particularly, double bax-/-bak-/- MEFs, we observed the inhibition of apoptosis. In conclusion, Olomoucine and Roscovitine caused apoptosis through an intrinsic pathway with Bax and Bak proteins being involved

7-Bromoindirubin-3′-oxime induces caspase-independent cell death

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3′-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3′-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3′-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin- 3′-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis. © 2006 Nature Publishing Group. All rights reserved